CNiFERs Cell-based Neurotransmitter Fluorescent Engineered Reporters

描述(由申请人提供)：我们建议推进一项技术，首次允许在体内检测任何与G蛋白偶联受体结合的神经递质。这包括单胺类物质，它们在神经调节中发挥基础作用，在成瘾和精神障碍中发挥生物医学作用，以及多肽递质，它们对神经内分泌系统以及血管张力和血流的控制非常重要。目前，只有有限的体内试验可用于检测单胺类和多肽类神经递质。这一缺陷是理解大脑中正常信号的主要障碍。为了填补这一缺失的空白，我们引入了基于细胞的神经递质荧光工程报告(CNiFERs)来光学测量体内外源受体的活性。CNiFER是一种克隆细胞系，它被设计成表达一种特定的代谢性受体，该受体偶联到G蛋白和一种基于FRET的遗传编码的钙传感器，该传感器检测细胞内[钙]+的变化。刺激代谢性受体导致胞浆[Ca~(2+)]升高，提供直接和快速的局部神经递质活性读数。CNiFERs被急性或慢性植入，并在体内用双光子显微镜测量荧光。这项新技术将使绘制体内信号的空间模式成为可能，空间精度高达100 5m，时间分辨率高达1 S。 与公共健康相关：Kleinfeld，David和Slesinger，Paul A.脑细胞之间的化学交流是动物运动并与环境互动的神经系统进行计算的基础。然而，对调节这种通讯的神经元递质和调制器的空间模式和时间释放的测量已经被证明是困难的。在这里，我们建议开发和实施一项新技术，CNiFERs(基于细胞的神经递质荧光工程报告程序)，可以与光学显微镜结合使用，作为一种手段来检测活体大脑中神经元和神经血管信号模式的模式。

DESCRIPTION (provided by applicant): We propose to advance a technology that, for the first time, will permit in vivo detection of any neurotransmitter that binds to a G-protein coupled receptor. This includes the monoamines, which play a fundamental role in neuromodulation and a biomedical role in addiction and mental disorders, and peptide transmitters, which are important for the neuroendocrine system and control of vascular tone and blood flow. At present, only limited in vivo assays are available to detect monoamines and peptide neurotransmission. This deficit is a major impediment to understanding normal signaling in the brain. To fill this gap of missing, we introduce Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for the optical measurement of exogenous receptor activity in vivo. A CNiFER is a clonal cell-line that is engineered to express a specific metabotropic receptor that couples to G proteins and a genetically encoded FRET-based Ca2+ sensor that detects changes in intracellular [Ca2+]. Stimulation of the metabotropic receptor leads to elevations in cytosolic [Ca2+], providing a direct and rapid readout of local neurotransmitter activity. CNiFERs are acutely or chronically implanted and fluorescence measured with in vivo two-photon microscopy. This new technology will make it possible to map the spatial patterns of in vivo signaling with up to ~ 100 5M spatial precision and ~ 1 s temporal resolution. PUBLIC HEALTH RELEVANCE: Kleinfeld, David and Slesinger, Paul A. Chemical communication between brain cells underlies the computations performed by a nervous system as animals' locomote and interact with their environment. Yet measurements of the spatial patterns and temporal release of the neuronal transmitters and modulators that mediate this communication have proven to be difficult. Here we propose to develop and implement a new technology, CNiFERs (cell-based neurotransmitter fluorescent engineered reporters), that may be used in conjunction with optical microscopy as a means to detect patterns of neuronal and neurovascular signaling patterns in the brain in vivo.